Smooth muscle and nonmuscle myosin II are regulated by phosphorylation of the 20 kDa regulatory light chain (RLC) located in the neck region. This region contains a single alpha-helical segment of the myosin heavy chain as well as the RLC and the essential light chain (ELC) and is called the regulatory domain. Previous studies have noted that single- headed species such as S-1 and single-headed myosin prepared by proteolysis of smooth muscle myosin have a high MgATPase activity regardless of the state of RLC phosphorylation. However, both of the species have a single regulatory domain. To determine whether interactions between adjacent regulatory domains of the two heads of myosin are essential for regulation, we have made a single-headed HMM- like molecule containing two regulatory domains using baculoviral expression of nonmuscle myosin IIB fragments. Sf9 cells were simultaneously infected with three viruses. One encoded an HMM-length fragment containing residues 1-1263. The second encoded residues 816- 1263 tagged at the C-terminus with the FLAG epitope. This fragment binds both the RLC and ELC and forms a coiled-coil dimer. The third virus expressed both the RLC and ELC. A combination of FLAG affinity column and ATP-dependent binding to actin was used to purify the heterodimeric single-headed molecule. Nondenaturing gels and rotary shadowing EM confirmed that a single-headed fragment had been prepared. Its steady- state actin-activated MgATPase was activated 3-7-fold by RLC phosphorylation. Since steady-state measurements typically underestimate the degree of myosin's regulation, we are currently using single-turnover kinetic experiments to determine the rate constant for product release in the off state.